Obesity, defined as an excess of body fat relative to lean body mass, is associated with important psychological and medical morbidities, the latter including hypertension, elevated blood lipids, and Type II or non-insulin-dependent diabetes mellitus (NIDDM). There are 6-10 million individuals with NIDDM in the U.S., including 18% of the population of 65 years of age (Kamel, H K, et al, Clin. Geriatr. Med., 1999, 15, 265). Approximately 45% of males and 70% of females with NIDDM are obese, and their diabetes is substantially improved or eliminated by weight reduction (Harris 1991, Diabetes Care, 14, 639).
Leptin, the product of the obese gene (Zhang, Y., et al. 1994, Nature 372, 425) functions as a peripheral signal to the brain that regulates food intake and energy metabolism. Leptin is thought to exert its action in the hypothalamus through its receptor, OB-R (Tartaglia, L. A., et al, 1995, Cell 83, 1263). Rodents with mutations that prevent normal expression of either leptin or full-length OB-R are profoundly obese, diabetic, and have a reduced metabolic rate (Coleman, D. L. 1978, Diabetologia 14, 141). However, human obesity does not appear to be associated with mutations in the genes encoding leptin or OB-R (Considine, R. V., et al. 1995, J. Clin. Invest. 95, 2986; Considine, R. V., et al, 1996, Diabetes 19, 992). Although mice with a mutant obese gene can be returned to normal weight by administration of recombinant leptin (Pelleymounter, M. A., et al. 1995, Science 269, 540; Halaas, J. L., et al, 1995, Science 269, 543; Campfield, L. A., et al, Science 269, 546), it seems unlikely that this approach will succeed in obese humans because their serum leptin levels are chronically elevated (Maffei, M., et al, 1995, Nature Med. 1, 1155; Considine, R. V., et al, 1996, N. Engl. J. Med. 334, 292; Sinha, M. K., et al, 1996, J. Clin. Invest. 98, 1277). Obese humans, therefore, appear to be “leptin resistant” (Maffei, M., et al, 1995, Nature Med. 1, 1155; Flier, J. S. & Elmquist, J. K. 1997, Nature Biotech. 15, 20; Campfield, L. A., et al, 1996, Horm. Metab. Res. 28, 619) in that they do not generate a signal commensurate with their serum leptin levels, perhaps because of defective transport of leptin across the blood-brain barrier (Caro, J. F., et al, 1996, Lancet 348, 159) or an inadequate OB-R response. Analysis of OB-R signaling pathways may reveal alternative therapeutic approaches of boosting OB-R responses to overcome leptin resistance and reverse obesity. Leptin and OB-R are members of the four-helical bundle cytokine and receptor superfamilies respectively (Tartaglia, L. A., et al, 1995, Cell 83, 1263; Madej, T., et al, 1995, FEBS Lett. 373, 13). OB-R is most closely related to the gp130 signal transducing receptor that is activated by cytokines such as Interleukin-6 and CNTF, whose signaling pathways have been intensively studied (Kishimoto, T., et al, 1992, Science 258, 593; Stahl, N. & Yancopoulos, G. D. 1994, J. Neurobiology 25, 1454). Leptin receptors are translated as several alternatively spliced products with different cytoplasmic domains (Tartaglia, L. A., et al, 1995, Cell 83, 1263; Lee, G.-H., et al, 1996, Nature 379, 632; Chen, H., et al, 1996, Cell 84, 491; Cioffi, J. A., et al, 1996, Nature Med. 2, 585; Wang, M. Y., et al, 1996, FEBS Lett. 392, 87), but only one isoform, known as the long form or OB-Rb, appears capable of mediating leptin's weight controlling effects (Lee, G.-H., et al, 1996, Nature 379, 632; Chen, H., et al, 1996, Cell 84, 491; Ghilardi, N., et al, 1996, Proc. Natl. Acad. Sci. USA 93, 6231; Baumann, H., et al, 1996, Proc. Natl. Acad. Sci. USA 93, 8374). Obese diabetic (db) mice have a mutation in OB-R that prevents expression of the long OB-R splice isoform, which renders them incapable of appropriately mediating leptin's actions (Lee, G.-H., et al, 1996, Nature 379, 632; Chen, H., et al, 1996, Cell 84, 491).
The discovery of new biologically active molecules, which are used as drugs for the treatment of life-threatening diseases, has involved two basic operations: (i) a more or less random choice of a molecular candidate, prepared either via chemical synthesis or isolated from natural sources, and (ii) the testing of the molecular candidate for the property or properties of interest. The discovery cycle is repeated indefinitely until a molecule possessing the desirable properties is located. In the majority of cases, the molecular types chosen for testing have belonged to rather narrowly defined chemical classes. For example, the discovery of new peptide hormones has involved work with peptides; the discovery of new therapeutic steroids has involved work with the steroid nucleus. As a result, the discovery of new functional molecules, being ad hoc in nature and relying predominantly on serendipity, has been an extremely time-consuming, laborious, unpredictable, and costly enterprise.
Modern theories of biological activity state that biological activities, and therefore physiological states, are the results of molecular recognition events. For example, nucleotides can form complementary base pairs so that complementary single-stranded molecules hybridize, resulting in double- or triple-helical structures that appear to be involved in regulation of gene expression. In another example, a biologically active molecule, referred to as a ligand, binds with another molecule, usually a macromolecule referred to as ligand-acceptor (e.g. a receptor or an enzyme), and this binding elicits a chain of molecular events which ultimately gives rise to a physiological state, e.g. normal cell growth and differentiation, abnormal cell growth leading to carcinogenesis, blood pressure regulation, nerve-impulse-generation and propagation, etc. The binding between ligand and ligand-acceptor is geometrically characteristic and extraordinarily specific, involving appropriate three-dimensional structural arrangements and chemical interactions.
A currently favored strategy for development of agents which can be used to treat diseases involves the discovery of forms of ligands of biological receptors, enzymes, or related macromolecules, which mimic such ligands and either boost (i.e., agonize) or suppress (i.e., antagonize) the activity elicited by the acceptor or receptor. The discovery of such desirable ligands has traditionally been carried out either by random screening of molecules (produced through chemical synthesis or isolated from natural sources, for example, see K. Nakanishi, Acta Pharm. Nord., 1992, 4, 319-328.), or by using so-called “rational” approach involving identification of lead-structure, usually the structure of the native ligand, and optimization of its properties through numerous cycles of structural redesign and biological testing (for example see Testa, B. & Kier, L. B. Med. Res. Rev. 1991, 11, 3548 and Rotstein, S. H. & Mureko, M. A., J. Med. Chem. 1993, 36, 1700). Since most useful drugs have been discovered not through the “rational” approach but through the screening of randomly chosen compounds, a hybrid approach to drug discovery has recently emerged which is based on the use of combinatorial chemistry to construct huge libraries of randomly-built chemical structures which are screened for specific biological activities. (Brenner, S. & Lerner, R. A. Proc. Natl. Acad. Sci. USA 1992, 89, 5381). Any screen of such huge libraries of randomly built chemical structures requires a cost-effective biological assay that is amenable to automation.
Leptin is traditionally assayed in vivo by injecting it to ob/ob mice and observing their reduction in body mass. This bioassay is cumbersome, takes a long time and not reproducible. It is also not suitable for e.g. high throughput screening of libraries containing millions of compounds. Yet, some simpler assay systems were described. For example, the finding that the long form of OB-R contains the sequence YXXQ (Tartaglia, L. A., et al, (1995) Cell 83, 1263), which is a motif that specifies STAT3 activation (Stahl, N., et al, (1995) Science 267, 1349), raised the possibility that STAT3 is critical for mediating leptin responses. Recent results verify that STAT3 is activated both in cultured cells (Ghilardi, N., et al, (1996) Proc. Natl. Acad. Sci. USA 93, 6931; Baumann, H., et al, (1996) Proc. Natl. Acad. Sci. USA 93, 8374; Rosenblum, C. I., et al, (1996) Endocrinology 137, 5178) and in vivo (Vaisse, C., et al, (1996) Nature Gen. 14, 95) by the long form of OB-R, and not by a truncated OB-R or the long form of OB-R with a mutant YXXQ motif (Baumann, H., et al, (1996) Proc. Natl. Acad. Sci. USA 93, 8374; White, D. W., et al, (1997) J. Biol. Chem. 272, 4065). PCT patent application No. WO9857177 proposes the use of STAT3 activation as a basis for a simple in vitro leptin bioassay. However, STAT3 is activated by many other hormones and cytokines, including IL-6, CNTF, interferon alpha and beta, growth hormone and many more cytokines and polypeptide hormones. Therefore, activation of STAT3 can not be used as a specific marker of leptin activity.
PCT application WO9740380 discloses the use of a DNA cassette termed “leptin response element”. Attachment of this DNA cassette to a promoter such as the thymidine kinase promoter and a reporter gene such as luciferase will provide a suitable reporter vector. Cells expressing the leptin receptor OB-Rb are transfected with the reporter vector. Such cells are proposed as tools for assay in vitro of leptin. However, according to PCT application WO9740380 the proposed “leptin response element” is identical with the “gamma activation sequence”, a well-known element that is activated by many other cytokines, and particularly interferon gamma, interferon alpha and interferon beta. Therefore, assays based on the gamma activation sequence will not be able to discriminate between leptin-mimetic activity and e.g., interferon-mimetic activity.
Still another in vitro assay is based on a chimeric receptor consisting of the extracellular domain of OB-R fused to a transmembrane domain and an intracellular domain of a reporter receptor such as the IL-3 receptor. It is shown that IL-3-dependent cells expressing said chimeric receptor can be used for measuring leptin, which promotes cell proliferation. Thus, upon exposure to leptin, said cells will proliferate and their proliferation may be determined by MTT staining or by incorporation of radiolabelled thymidine (Verploegen S A, et al, 1997, FEBS Lett. 405, 237). This assay system is reliable, it is useful for measuring leptin and is amenable to high throughput screening. However, molecules selected by this high throughput screening based on this assay may not be leptin-mimetic at all. It was shown for instance that a true insulin mimetic agent, found by a high throughput screen acts by interacting with the cytoplasmic domain of the insulin receptor (Zhang B., et al, 1999, Science 284, 974). Therefore, there is a risk that molecules selected by chimeric receptors such as said leptin-IL-3 receptor chimera will interact with the cytoplasmic IL-3-derived domain of the chimera and therefore will be IL-3 mimetic rather than being leptin mimetic. Hence this assay is not reliable for screening of leptin-mimetic agents.
Leptin, the product of the obese gene is expressed in adipocytes and it regulates food intake and energy metabolism through its hypothalamic receptor OB-Rb. Lack of leptin, as in the case of ob/ob mice or lack of OB-Rb, as in the case of db/db mice leads to morbid obesity. However, the most common cases of human obesity are not associated with leptin deficiency. In fact, serum leptin correlates with body mass index and therefore obese individuals have high levels of serum leptin. This correlation led to the notion that obesity is associated with some form of leptin resistance. One possible mechanism of leptin resistance is the inefficient transfer of leptin through the blood brain barrier. Indeed, intracerebroventricular administration of leptin was significantly more effective in reducing adipose tissue mass of rodents as compared with peripheral routes of leptin administration.
Development of leptin-mimetic agents that may cross the blood brain barrier may solve the problem of leptin resistance. For this purpose it is advantageous to screen libraries consisting of low molecular weight agents in order to identify individual agents that exhibit leptin-mimetic activity. Such a screen requires a simple and specific bioassay of leptin activity. So far, as described above, the biological activity of leptin could be determined only by assay in animals. Such assays are cumbersome and therefore not suitable for screening libraries consisting of millions of different substances. Several in vitro assays were described but they are not specific for leptin. Therefore, there is a need to establish a simple and specific assay of leptin's activity that will be easily amenable to automation.